【摘要】 为培育出鸭短喙矮小综合征病毒（SBDSV）弱毒株并评价其免疫效果，本研究将强毒SBDSV M15株在番鸭胚成纤维细胞（MDEF）中连续传95代（C95），每代均观察细胞病变（CPE）。结果显示，C1～C4代病毒接种的细胞均无CPE，从C5代开始出现CPE；自C50代以后，每代细胞出现CPE达80%的时间由接种后7 d逐渐缩短为5 d。采用《中国兽药典》附录中方法检测每隔10代病毒的纯净性；将每隔10代的病毒感染MDEF，通过间接免疫荧光试验（IFA）鉴定各代次病毒及测定各代次的病毒滴度(TCID₅₀)；将各代次病毒分别以不同剂量接种2日龄易感半番鸭，在21 d观察期内观察并记录各组半番鸭的临床症状、计算发病率及死亡率，于观察期结束后，对各组半番鸭称重并测量其喙长、喙宽，计算其喙长宽比等生长发育指标，评估各代次病毒对半番鸭的致病性。纯净性检测结果显示：检测的各代次病毒均无细菌、霉菌、支原体和外源病毒污染；IFA结果显示，感染不同代次SBDSV的MDEF均能被鹅细小病毒单克隆抗体（GPV MAb）识别，出现亮绿色特异性荧光，而阴性对照和其他相关病毒MAb作为一抗的MDEF中均无绿色荧光；病毒滴度测定及致病性试验结果显示，随着传代次数的增加，SBDSV的滴度逐渐升高(10^2.5TCID₅₀/0.1 mL～10^5.5TCID₅₀/0.1 mL)且趋于稳定的10^5.5TCID₅₀/0.1 mL;C10、C30和C50代病毒对雏鸭的致病性均较强，雏鸭均出现了一定的临床症状，生长发育受到影响，且雏鸭的发病率分别为87.5%（7/8）、37.5%（3/8）和25%（2/8），但均无死亡；C70代病毒组仅一只鸭出现体重略轻的症状，至C80代已失去对雏鸭的致病性，将致弱病毒命名为SBDSV MA株，将C80代致弱病毒命名为MA0株。将不同代次的致弱病毒MA0～MA15株均以5×10^5.5TCID₅₀分别接种2日龄半番鸭和樱桃谷鸭，观察21 d，评估致弱病毒的安全性。将上述不同代次的致弱病毒均以4×10³TCID₅₀接种2日龄半番鸭，7 d后采血测定各组鸭血清中的中和抗体效价；同时各免疫组以5×10⁴TCID₅₀的强毒SBDSV M15株攻毒，观察14 d，评估致弱病毒的免疫保护效果。将MA5代病毒在雏半番鸭体内连续盲传5代，评估该病毒的遗传稳定性。结果显示，不同代次致弱病毒（MA0、MA5、MA10、MA15）接种的两种雏鸭均无临床症状且生长发育均良好；各代次致弱病毒接种后7 d全部鸭血清均出现中和抗体，平均抗体效价在3.0Log2以上；攻毒后14 d，各免疫组鸭均无任何临床症状且生长发育良好，与阴性对照组鸭基本一致，且致弱病毒对雏鸭的免疫保护率均达100%;MA5代致弱病毒在雏鸭体内连续盲传5代，每代雏鸭均无任何临床症状，生长发育均正常，且各组实验鸭各器官均未出现剖检病变。上述结果表明，不同代次的SBDSV致弱株均纯净、安全性好、免疫原性和遗传稳定性较强，可作为SBDS弱毒疫苗的候选株，本研究为SBDS活疫苗的研制奠定了良好实验基础。更多还原

【Abstract】 To develop an attenuated strain of duckling short beak and dwarfism syndrome virus （SBDSV） and evaluate its immunogenic potential,we passaged the SBDSV strong strain M15 in Muscovy duck embryo fibroblasts （MDEF） for 95 generation（C95）,and monitoring the cytopathic effect （CPE） in each generation.The results showed that C1 to C4 generations of virus-infected cells did not show CPE,while C5 generation appeared CPE.From C50 generation onwards,the time it took for CPE to reach 80%of the cells reduced from 7 days after infection to 5 days.To ensure viral purity,we tested every 10th generation using the methods outlined from the Chinese Pharmacopoeia.Each 10th generation virus was infected with MDEF,and the virus was further characterized by indirect immunofluorescence assay （IFA） and titered using 50%tissue culture infective dose (TCID₅₀).Each generation of virus was inoculated into 2-day-old susceptible mule duckling at different doses to track clinical symptoms,morbidity and mortality over 21 days,with growth metrics such as weight,beak length and width to evaluate the pathogenicity of each generation of virus.The results showed that the viruses from each generation were free from bacterial,mold,mycoplasma,and exogenous virus contamination.IFA results showed that specific bright green fluorescence in MDEF infected with different generations of SBDSV using goose parvovirus monoclonal antibody （GPV MAb）,with no green fluorescence in the negative control or with other related viral MAbs.Titer determination and pathogenicity tests revealed that as the number of passages increased,the SBDSV titer gradually rose from 10^2.5TCID₅₀/0.1mL to 10^5.5TCID₅₀/0.1mL and stabilized at 10^5.5TCID₅₀/0.1mL.The C10,C30,and C50generations exhibited strong pathogenicity,with infection rates of 87.5%（7/8）,37.5%（3/8）,and 25%（2/8）,respectively,but no deaths were observed.By the C70 generation,only one duckling showed slightly reduced weight,and by C80 generation,the virus had lost its pathogenicity,named the attenuated viral was MA strain,Naming the C80 attenuated virus strain MA0.We assessed the safety of different generation of attenuated viruses （MA0-MA15 strains） by inoculating 2-day-old mule ducklings and Cherry Valley ducklings with 5×10^5.5TCID₅₀of each strain and monitoring for 21 days.The blood serum neutralizing antibody titers of the different attenuated viruses were determined by inoculating 2-day-old mule ducklings with 4×10³TCID₅₀of each strain and collecting blood7 days later.Additionally,each immunized group was challenged with 5×10⁴TCID₅₀of the strong SBDSV M15 strain and observed for 14 days to evaluate the immune protection effect of the attenuated virus.Genetic stability of the MA5 generation virus was evaluated by blind passage in ducklings for 5 generations.The results showed that the ducklings inoculated with different generation attenuated viruses （MA0,MA5,MA10,MA15） displayed no clinical symptoms and exhibited healthy growth and development.All the blood serum of the ducklings inoculated with the different generation attenuated viruses appeared neutralizing antibody 7 days after inoculation,with the average antibody titer above 3.0Log2.After the challenge,all immunized ducklings remained symptomfree and developed well,matching the negative control group,with a 100%immune protection rate.The MA5 generation of attenuating virus was blindly transmitted in ducklings for 5 consecutive generations,and each generation of ducklings showed no clinical symptoms,normal growth and development,and no autopsy lesions in any organ of the experimental ducks in each group The above results indicate that all the attenuated viruses strains of SBDSV from different generations are pure,safe,highly immunogenicity and genetically stable,and making them suitable candidate strains for an attenuated SBDSV vaccine.These results provide a solid experimental foundation for the development of live SBDSV vaccines.更多还原